Type-it Fast SNP Probe PCR Kit

For accurate and reliable SNP genotyping using TaqMan® or TaqMan® MGB probes

Validated using TaqMan® SNP Genotyping Assays

Automated allele calling and tight fluorescence clusters

Suitable for difficult SNP loci or very low template amounts

Up to 40% time savings due to fast cycling procedure

The Type-it Fast SNP Probe PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a newly developed buffer system, both of which enable reliable and clear allelic discrimination. The combination of all components provided in the master mix result in improved accuracy and highly specific probe binding. The Type-it Fast SNP Probe PCR Kit is validated using TaqMan® SNP Genotyping Assays and enables reproducible SNP genotyping — even with difficult SNP loci (e.g., GC rich) or low amounts of starting template.

The Type-it Fast SNP Probe PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Successful automated allele calling.

Average automated allele call rates for a panel of different DNAs were analyzed using 6 different TaqMan® MGB-based SNP genotyping assays and 1 ng of genomic DNA from each of the 60 samples. PCR was performed with the indicated products in 384-well plates in a 5 μl reaction volume. Allelic discrimination plate read was performed on an Applied Biosystems 7900HT instrument. The Type-it Fast SNP Probe PCR Kit consistently resulted in the highest call rates and the lowest error rates.

Highly specific probe binding.

Buffer systems from other suppliers used for SNP genotyping often show poorer separation of allele clusters due to nonspecific binding of the mismatch probe. The Type-it Fast SNP Probe Buffer System provides increased discrimination by narrowing the probe melting temperature window, thereby reducing nonspecific binding of the mismatch probe. Wide and clear separation of allele clusters is obtained.

Allelic discrimination was performed with a panel of 80 different genomic DNAs (10 ng or 1 ng each in 5 μl reactions) using either the [A] Type-it Fast SNP Probe PCR Kit or [B] a genotyping master mix from Supplier A. Even when using low amounts of template, the Type-it Fast SNP Probe PCR Master Mix outperformed the master mix from Supplier A and provided tight clustering of alleles and reliable results. PCR was performed with a TaqMan® SNP genotyping assay for rs 951134 (aryl-hydrocarbon receptor repressor gene) and 2 no template controls (NTCs) and analyzed on an Applied Biosystems 7900HT instrument. Black: NTCs; blue: homozygous for T allele (FAM fluorescence); green: heterozygous samples; red: homozygous for A allele (VIC fluorescence). Data was analyzed with 98% confidence.

High specificity and sensitivity

HotStarTaq Plus DNA Polymerase, provided in the master mix, which ensures highly specific amplification, even with low template amounts. HotStarTaq Plus DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer dimers at low temperatures.

Unique buffer system

The Type-it Fast SNP PCR Buffer, also included in the master mix, is specifically designed for fast-cycling SNP genotyping using sequence specific 5'-nuclease probes. The unique composition of the Type-it Fast SNP PCR Buffer ensures highly stringent and specific binding of the allele-specific probe (match probe). This is due to the altered melting behaviour of the probes resulting in a narrower probe melting temperature window. Based on the original QIAGEN PCR Buffer, this innovative formulation enables a high ratio of specific-to-nonspecific primer binding during the short annealing step of every PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced and often not required.

Additives in the Type-it Fast SNP PCR buffer, such as Q-Solution, provide reaction conditions for amplification of difficult genomic regions and difficult SNP loci. Innovative Q-Bond technology (for fast cycling) allows SNP genotyping results to be achieved faster (see figure "Mechanism of fast cycling during annealing"). The master mix also contains ROX dye at a concentration compatible with SNP genotyping on all instruments from Applied Biosystems. However, ROX dye is also compatible with other instruments not requiring ROX as a passive reference dye (see table).

Kit features

Kit contents

Features

2x Master Mix format*

Developed for SNP genotyping with TaqMan® MGB probes
Suitable for use with all cyclers suitable for SNP genotyping

The Type-it Fast SNP Probe PCR Kit is functionally verified with commercially available SNP genotyping assays and is compatible with TaqMan® MGB Probes, as well as user-developed probe-based assays consisting of TaqMan® MGB, TaqMan®, or other dual-labeled probes. The kit includes streamlined, preoptimized protocols for fast and reliable analysis.

Fast-cycling procedure for straightforward results

The Type-it Fast SNP Probe PCR Master Mix provides reaction conditions for fast-cycling PCR using sequence-specific probes. The buffer includes proprietary Q-Bond Molecule, which allows short cycling times on standard cyclers and on fast cyclers with rapid ramping rates. The fast-cycling procedure increases throughput by reducing PCR run times by up to 40% (see figure "Genotyping workflow").

Convenient kit format

The kit is provided in a ready-to-use, preoptimized master mix format for greater convenience. Use of a master mix saves time, simplifies handling for reaction setup, and increases reproducibility by eliminating many possible sources of pipetting errors and contamination — pipetting steps are minimized and tedious calculations are eliminated. HotStarTaq Plus DNA Polymerase (included in the master mix) is activated by a 5-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. Room-temperature reaction setup using the master mix is fast and easy.